Bacteria-based surface contamination on biomaterials, medical devices and other surfaces such as food processing plants, drinking water systems; marine installations, textiles etc. result in millions of death every year worldwide accompanied by huge financial loss. In order to prevent the surface based contamination, the use of antimicrobial polymers has attracted significant attention in recent years. The modification of surfaces using antimicrobial polymers prevents or retards the surface bacterial contamination of biomaterials and other devices with the possibility of reduced surface migration of antimicrobial agents resulting in reduced environmental pollution and toxicity. As one of the most effective biocides, N-halamine based polymers to prevent surface contamination have gained a great deal of interest in recent years. In this thesis, several new cyclic N-halamine based vinyl monomers with enhanced halogen binding capacity were synthesized and copolymerized with other vinyl monomers such as methyl methacrylate (MMA) and styrene and their ability to prevent bacterial adhesion and colonization were evaluated by coating these
copolymers on various surfaces.
In Chapter 1, a general introduction of bacteria, bacterial biofilm and the stages involved in the development of biofilm on surfaces are discussed. The parameters that influence the biofilm growth on surfaces are also discussed. An overview on various kinds of surfaces available for biofilm growth is presented. A comprehensive survey of different kinds of antimicrobial polymers including N-halamines in preventing surface-based contamination has been made. The development and application of hydantoin-based antibacterial polymers with improved halogen binding sites has been proposed as the major objective of the present work.
Chapter 2 deals with the synthesis, characterization and antibacterial evaluation of a new hydantoin based polymer. Initially, a new polymerizable vinyl hydantoin monomer, 4-(2- (2,5-dioxoimidazolidin-4-yl)-acetamido)phenyl methacrylate containing an additional amide group, in addition to the amide and imide functions that exist in hydantoin, was synthesized to increase the halogenation capacity and consequently its antimicrobial properties. The synthesized monomer was copolymerized with commercially available monomers such as MMA or styrene by free radical polymerization. The monomer and its copolymer were characterized using NMR, FTIR, HRMS, XPS and TG-DSC. Glass surfaces were modified with the copolymers by employing the spin-coating method. The modified glass surfaces were treated with aqueous bleach to activate the chloramide functions. Further, the modified surface was challenged with Gram positive S. aureus and Gram negative E. coli bacteria to evaluate the antimicrobial efficacy. The coated surfaces exhibited total kill of both strains in 15 min of exposure time. However, a decrease in chlorine content was observed in the polymer during the durability and rechargeability studies which was attributed to the presence of the vicinal C–H group (tertiary hydrogen) next to N–Cl in the hydantoin ring triggering the release of HCl via the elimination reaction. The released HCl was also found to be responsible for the acid catalyzed hydrolysis of ester to compromise the durability, rechargeability and antibacterial activity of polymer in long term use. To overcome these issues, a new strategy was adopted to synthesize a hydantoin monomer that would resist the HCl elimination and is the subject of discussion in Chapter 3.
Chapter 3 focuses on the development of new hydantoin monomer with improved rechageability, durability and antibacterial activity. The Knoevenagel condensation method was employed to remove vicinal C-H group next to N-Cl in the hydantoin ring to avoid the elimination of HCl from the polymer. This enhanced the stability of the N–Cl bond in the hydantoin ring and contributed to the improved durability and better antimicrobial activity of the polymer. The synthesized monomer was copolymerized with MMA and styrene through free radical polymerization. The copolymers were spin-coated onto the glass surface and treated with aqueous bleach to charge the surface by generating the chloramide function. The activated surface was exposed to both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. The surface modified with the copolymer containing 5 mole percent (mol%) hydantoin monomer exhibited total kill in 20 min of exposure, whereas the polymer containing 10 mol% hydantoin monomer showed total kill in 13 and 15 min exposure against S. aureus and E. coli, respectively. The durability and rechargeability studies of the polymer showed excellent stability against multiple washings cycles.
Chapter 4 deals with functionalization of stainless steel surface with new hydantoin based polymer through covalent metal-oxane bond formation. Initially, a new hydantoin monomer, (Z)- N-((4-(2,5-dioxoimidazolidin-4ylidene)methyl)phenyl)methacrylamide (DMPM), containing three halogen binding sites was synthesized and copolymerized with 3-(methacryloyloxy)propyl trimethoxysilane) in the presence of benzyl peroxide as initiator. The monomer and its copolymer were characterized using NMR, FTIR, HRMS, XPS and TG-DSC. The stainless steel surface was oxidized with piranha solution to covalently immobilize the copolymer on the surface. The modified surface was challenged for antibacterial and anti-biofilm activity. The modified surface exhibited total kill of bacteria such as S. aureus and E. coli in 10 and 12 min respectively. The anti-biofilm activity of the modified surface evaluated using a combination of fluorescence-based metabolic activity and scanning electron microscopy imaging suggested the comprehensive damage of S. aureus and E. coli biofilm architecture.
In Chapter 5, the potential for water decontamination using polymeric microspheres derived from hydantoin monomer is discussed. Copolymer microspheres of DMPM with MMA using ethyleneglycol dimethacrylate (EGDM) as a crosslinking agent via free radical polymerization were synthesizd. Further, to improve the antibacterial activity, microspheres with rough surface were fabricated using toluene during polymerization to enhance the surface area of the microspheres. Microspheres were characterized using different techniques such as SEM, NMR, FTIR, XPS, and Brunauer–Emmett–Teller (BET) adsorption analysis. Both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria were selected to test the bactericidal efficacy of these microspheres. BET analysis suggested a significant increase in surface area of microspheres prepared with toluene. These rough surface microspheres exhibited enhanced antibacterial activity compared to microspheres with a smooth surface. To demonstrate the water purification potential of the microspheres, a glass column was packed with chlorinated polymeric microspheres and challenged with contaminated water. The microspheres exhibited potential antibacterial activity. The chlorinated polymeric microspheres were found to be non-toxic to the fibroblast cells thereby making the microspheres suitable for water purification.
Publication:
[1] R.K. Rai, A. Jayakrishnan. Synthesis and polymerization of a new hydantoin monomer with three halogen binding sites for developing highly antibacterial surfaces New J. Chem., 2018, 42, 12152-12161.
[2] R.K. Rai, A. Jayakrishnan. Development of new hydantoin-based biocidal polymers with improved rechargeability and anti-microbial activity”, New J. Chem., 2019, 43, 3778-3787.
[3] R.K Rai, H. Kanniyappan, V. Muthuvijayan, K. Venkitasamy and A. Jayakrishnan, "Durable polymeric N-halamine functionalized stainless steel surface for improved antibacterial and anti-biofilm activity." Mater Adv, 2(3):1090-98, 2021..
[4] V. Ravichandran, R.K Rai, V. Kesavan, and A. Jayakrishnan Tyrosine-derived novel antimicrobial hydantoin polymers: Synthesis and evaluation of antibacterial activities. J. Biomater. Sci. Polym. Ed 18, 2131-2142, 2017.